Genome-wide investigation of SQUAMOSA promoter binding protein-like genes in Liriodendron and functional characterization of LcSPL2

Abstract The plant-specific SQUAMOSA promoter-binding protein-like (SPL) transcription factors play a pivotal role in various developmental processes, including leaf morphogenesis and vegetative to reproductive phase transition. Liriodendron chinense and Liriodendron tulipifera are widely used in landscaping due to their tulip-like flowers and peculiar leaves. However, the SPL gene family in Liriodendron has not been identified and systematically characterized. We systematically identified and characterized the SPL family members in Liriodendron, including phylogeny, gene structure and syntenic analyses. Subsequently, we quantified the expression patterns of LcSPLs across various tissue sites through transcription-quantitative polymerase chain reaction (RT-qPCR) assays and identified the target gene, LcSPL2. Finally, we characterized the functions of LcSPL2 via ectopic transformation. Altogether, 17 LcSPL and 18 LtSPL genes were genome-widely identified in L. chinense and L. tulipifera, respectively. All the 35 SPLs were grouped into 9 clades. Both species had three SPL gene pairs arising from segmental duplication events, and the LcSPLs displayed high collinearity with the L. tulipifera genome. RT-qPCR assays showed that SPL genes were differentially expressed in different tissues, especially. Because LcSPL2 is highly expressed in pistils and leaves, it was selected to describe the SPL gene family of L. chinense by ectopic expression. We showed that overexpression of LcSPL2 in Arabidopsis thaliana resulted in earlier flowering and fewer rosette leaves. Moreover, we observed that overexpression of LcSPL2 in A. thaliana up-regulated the expression levels of four genes related to flower development. This study identified SPL genes in Liriodendron and characterized the function of LcSPL2 in advancing flower development.


Introduction
Transcription factors (TFs) play crucial roles in plant growth and development.TFs from diverse families, such as HOMEODOMAIN-LEUCINE ZIPPER (HD-Zip), R2R3-MYB, AP2/ERF, MADS-box and SQUAMOSA promoterbinding protein-like (SPL), have been identified and documented in previous studies (Fu et al. 2012;Liu et al. 2021;Yang et al. 2021b;Zong et al. 2021;Tu et al. 2022).These TFs are recognized for their involvement in leaf and flower development.SPL proteins constitute a class of plantspecific TFs characterized by a highly conserved SBP domain consisting of 76 amino acid residues (Birkenbihl et al. 2005).This domain contains two zinc finger structures, one being C3H and the other being C2HC, along with a nuclear localization signal (NLS) (Klein et al. 1996;Yamasaki et al. 2004;Birkenbihl et al. 2005).Notably, the second zinc finger partially overlaps with the NLS, located at the C-terminal of the SBP domain, guiding SPL proteins into the nucleus to regulate the expression of target genes (Birkenbihl et al. 2005;Yamasaki et al. 2006).Among the first two SBP domain proteins reported in Antirrhinum majus, AmSBP1 and AmSBP2 were found to bind to the promoter of SQUAMOSA, thereby regulating early flower development (Klein et al. 1996).Since then, SPL proteins have been extensively identified and characterized in various plant species, including Arabidopsis thaliana, rice (Oryza sativa), poplar (Populus trichocarpa), maize (Zea mays), Medicago truncatula, Codonopsis pilosula, Populus euphratica and Carya illinoinensis (Moreno et al. 1997;Cardon et al. 1999;Xie et al. 2006;Li and Lu 2014;Wang et al. 2019Wang et al. , 2021;;Yang et al. 2021a).
SPLs have been extensively studied in both model and nonmodel plants, underscoring their pivotal regulatory roles in various aspects of plant growth and development.These roles encompass controlling plastochron length, orchestrating the transition from vegetative to reproductive phases, determining the timing of flowering, regulating plant fertility, influencing trichome distribution, modulating lateral root development, governing floral organ development and shaping leaf morphology (Wang et al. 2008(Wang et al. , 2009(Wang et al. , 2016;;Yamaguchi et al. 2009;Xing et al. 2010;Yu et al. 2010Yu et al. , 2015;;Jung et al. 2012Jung et al. , 2016;;Gao et al. 2018a;Cao et al. 2019).Furthermore, SPLs can be categorized into two groups: microRNA-targeted SPLs and non-microRNA-targeted SPLs (Xu et al. 2016;He et al. 2018;Zheng et al. 2019).Among them, microRNA156 has emerged as a major regulatory factor in the vegetative phase transitions of A. thaliana and other flowering plants, establishing the miR156-SPL pathway as a novel mechanism for age-dependent flowering regulation (Bergonzi et al. 2013;He et al. 2018).Previous studies have shown that miR156 levels are higher during the juvenile phase and significantly decrease in the adult phase, leading to an age-dependent up-regulation of SPL expression levels targeted by miR156.This up-regulation initiates the transition to the reproductive stage (Wang et al. 2009;Wang 2014).Crucially, the overexpression of miR156 negatively regulates several SPL genes and postpones the transition from the vegetative to reproductive stages, indicating that SPLs act as ageing genes, and SPLs overexpression promotes the transition from juvenile to adult plants (Shalom et al. 2015;He et al. 2018;De Paola et al. 2023).However, the functions of SPLs and the miR156-SPL module in Liriodendron plants have not been fully explored and remain unknown.Therefore, further research is necessary to investigate their roles in the regulation of growth and development in Liriodendron plants.
The Liriodendron genus, belonging to the Magnoliaceae family, comprises two species such as L. chinense and L. tulipifera (Chen et al. 2018).Both species are renowned for their exceptional wood quality and captivating appearance, particularly their tulip-like flowers.These flowers not only bestow upon them high ornamental value but also play a crucial role in their reproduction.However, the extensive geographical distribution of these species results in variations in their flowering times (Hao et al. 1995;Li et al. 2007), and the prolonged juvenile phase hinders the process of genetic improvement and their widespread utilization.Therefore, investigating the mechanism of flower induction and development, and abbreviating the juvenile phase in Liriodendron is of paramount importance.
In this study, we identified 17 LcSPL and 18 LtSPL genes based on the reference genomes of L. chinense (Chen et al. 2018) and L. tulipifera (YP108A v1.1, DOE-JGI, http:// phytozome-next.jgi.doe.gov/).A comprehensive analysis was performed on these genes, including an investigation of their sequence composition, gene structure, cis-acting elements and collinearity.Additionally, we examined the evolutionary relationships of LcSPL genes across several species, including L. chinense, L. tulipifera, M. truncatula, poplar, A. thaliana and rice.Furthermore, the tissue expression profiles of LcSPLs in various tissues of L. chinense were investigated.To gain insights into the functional role of LcSPL2 in flower development, we overexpressed LcSPL2 in A. thaliana and analysed the phenotypes of the transgenic plants.These findings provide a solid foundation for further characterizing the SPL genes in L. chinense.

Plant materials
Samples of root, stem, leaf, sepal, flower bud, petal, pistil and stamen from L. chinense were collected from a provenance plantation in Jurong County, Zhenjiang City, Jiangsu Province (32°7ʹN, 119°13ʹE).Each sample was replicated three times for robustness.
For the A. thaliana experiments, both wild-type (WT) and transgenic A. thaliana plants of the Colombia ecotype (Col-0) background were used.The seeds were sown on 1/2 MS solid medium and 1/2 MS solid medium supplemented with 30 mg/L Hygromycin B, respectively, and then placed in the dark at 4 °C for 48 h.Subsequently, the seedlings were transferred to an artificial illumination incubator set at long-day conditions (16 h of light, 8 h of dark) with a temperature of 23 °C, humidity of 70 % and a light intensity of 130-150 μmol m −2 s −1 .

Identification and physicochemical properties analysis of LcSPL proteins
To identify SPL proteins from the whole genome of L. chinense and L. tulipifera, first, 16 A. thaliana SPL proteins (downloaded from the Plant Transcription Factor Database) were used to query sequences to blast the L. chinense and L. tulipifera protein data by performing a local BLASTP alignment with an E value of 1 × 10 −5 (Chen et al. 2018).Then we used the hidden Markov Model (HMM) of SBP domain (PF03110) (downloaded from Pfam database (http://pfam.xfam.org/family/pf03110) to align the two types protein data by using the HMMER software (Potter et al. 2018)).The two types of proteins that appeared in both BLASTP alignment and HMM search results were considered as candidate LcSPL and LtSPL proteins.These SPL proteins were aligned to the Pfam database to remove proteins without the SBP domain, and the remaining proteins were identified as LcSPL proteins.Then, we used the ProtParam tool (https://web.expasy.org/protparam/) to analyse the physicochemical properties of these LcSPLs and LtSPLs.

Phylogenetic analysis, multiple sequence alignments and conserved motif identification of LcSPL proteins
Phylogenetic trees comparing L. chinense, L. tulipifera, M. truncatula, popular, A. thaliana and rice (Cardon et al. 1999;Xie et al. 2006;Li and Lu 2014;Wang et al. 2019) were constructed with the Neighbor-Joining (NJ) method, and the specific parameters were Poisson model and 1000 bootstrap replications by using the MEGA software (Hall 2013).Then, we used the MEME tool (https://meme-suite.org/meme/tools/ meme) to identify conserved motifs in LcSPL and LtSPL proteins (the number of conserved motifs was set as 10), and sequences of identified conserved motifs were aligned to Pfam database to confirm their corresponding domains.

Synteny analysis, chromosome localization and cisregulatory elements identification of LcSPL genes
Intraspecific synteny and interspecific synteny analysis were performed through the methods described in our previous study (Tu et al. 2022).The genome data of A. thaliana and poplar that were used for interspecific synteny analysis were downloaded from Phytozome 13 database (https:// phytozome-next.jgi.doe.gov/).The synteny analysis results were visualized by using the Advanced Circos tool.
To identify cis-elements, we analysed the 2000-bp sequences upstream of the translation start site for each LcSPLs and LtSPLs.Utilizing the PlantCare online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)with default parameters, we identified predicted cis-regulatory elements present in the gene promoters.For the determination of the gene structure and chromosome localization of LcSPLs and LtSPLs, a local BLASTN search was performed.In this search, the sequences of LcSPLs and LtSPLs were aligned to the genomes of L. chinense and L. tulipifera.Zhang et al. -Genome-wide investigation of SPL genes in L. chinense

RNA extraction and detection of gene expression level
The RNA Prep Pure Plant Kit (Tiangen, Beijing, China) and Evo M-MLV RT Premix for qPCR (AG11706, Accurate Biotechnology, Hunan Co, Ltd.) were employed for the extraction of total RNA from L. chinense samples and subsequent cDNA synthesis, following the respective instructions.Total RNA from WT A. thaliana and transgenic plants was extracted using the KK Fast Plant Total RNA Kit (ZP405K, ZOMANBIO, Beijing).Subsequently, the Evo M-MLV RT Premix for RT-qPCR was utilized for cDNA synthesis.
We used RT-qPCR to detect the expression level of LcSPLs.A total of 10 μL reaction volume included 10 μM forward primer, 10 μM reverse primer, 0.2 μL 50× ROX, 5 μL 2× SYBR ® Green Pro Taq HS Premix (provided by SYBR ® Green Premix Pro Taq HS qPCR Kit (AG11701, ACCURATE BIOTECHNOLOGY)), 1 μL cDNA and up to 10 μL RNasefree water.For the amplification process and method of data analysis, refer to our previous study (Tu et al. 2022).LcACT97 was used as internal control gene for L. chinense, and AtACT2 was used as a reference gene for A. thaliana.

Ectopic transformation of LcSPL2 in A. thaliana and phenotypic analyses
For constructing the LcSPL2 overexpression vector, the coding sequence (CDS) of LcSPL2 was inserted into the pCAMBIA 1300 vector at its cleavage sites using KpnI and XbaI restriction endonucleases (TaKaRa, Dalian, China).The recombinant plasmids, confirmed by sequencing, were transferred to Agrobacterium tumefaciens strain GV3101 through a heat-shock method and then the Agrobacterium-mediated floral dip method was used for the A. thaliana genetic transformation.T3 generation homozygous transgenic lines were selected for further analyses.The initial flowering time and the number of rosette leaves of both WT and transgenic A. thaliana plants were recorded.Subsequently, samples were collected from both WT and transgenic A. thaliana plants when the transgenic A. thaliana first blossomed.Each line consisted of at least 10 seedlings for phenotype observation.All the samples were immediately frozen and stored at −80 °C until use.
RT-qPCR was used to quantitatively assess the transcription levels of LcSPLs in various tissues of L. chinense.Additionally, the expression levels of flower developmentrelated genes (AtSOC1, AtFT, AtFUL and AtAP1) were analysed in transgenic A. thaliana.LcActin97 was utilized as the reference gene for L. chinense, while AtActin2 served as the reference gene for A. thaliana.All primer sequences are provided in Supplementary Material 2.

Data analysis
We used the 2 −ΔΔCT method to calculate the relative expression levels of LcSPLs.All RT-qPCR data were analysed with IBM SPSS Statistics (Version 25; SPSS, Inc., Chicago, IL, USA) and GraphPad Prism (version 8.0.0 for Windows, GraphPad Software, San Diego, CA, USA), the statistical validity was analysed by one-way analysis of variance (ANOVA).Duncan's multiple range test was applied.Differences were considered significant when P < 0.01 or P < 0.05.All heatmaps were constructed with the TBtools software (https://github.com/CJ-Chen/TBtools/releases).

Identification of SPL genes in L. chinense and L. tulipifera
In this study, a total of 35 SPL genes were identified in the genus Liriodendron.Specifically, 17 SPLs in L. chinense were denoted as LcSPL1a to LcSPL15 (Table 1), while the 18 SPLs in L. tulipifera were designated as LtSPL1a to LtSPL15 (Table 2) based on their closest orthologs in A. thaliana.The predicted LcSPL and LtSPL proteins exhibited considerable variability in terms of molecular weight and length.
In the phylogenetic tree, the 121 SPLs were classified into 9 clades, denoted as G1-G9.Notably, the number of SPLs varied among the clades, with the G5 clade containing the highest number of SPLs from Liriodendron, while the other 8 clades consisted of 1-2 SPLs each.It is interesting to observe that each member of the LtSPLs, except for LtSPL6, had a homologous counterpart in the LcSPLs.Within the G2 clade, SPL proteins from the same species clustered together in clades, indicating higher conservation of SPLs within species.Additionally, the G4 and G6 clades contained fewer species compared with the remaining clades, and AtSPLs were not found in the G4 clade.

Gene structure, conserved domains and motifs of the LcSPLs and LtSPLs
To gain insights into the sequence characteristics of SPL genes in Liriodendron, we conducted a systematic analysis of gene structure, conserved domains and motifs.The gene structure analysis revealed that genes within the same evolutionary clade shared an identical number of exons (Tables 1 and  2).Similarly, SPL proteins within the same clade exhibited conserved structural domains and motifs (Fig. 2).Notably, the G5 clade displayed a more intricate structure compared with other clades, containing an additional conserved structural domain, Ank2, along with motifs 4, 5 and 8, which were unique to G5.
Upon further comparison, it has been determined that all SPL proteins shared the SBP structural domain, comprising two zinc finger structures (Zn-1 and Zn-2) and an NLS.However, specific variations were noted at specific sites of the SBP domain (Fig. 3).For instance, the L residue was exclusively identified at the second site of the SBP domain in G1 members (LcSPL13a and LtSPL13a, LcSPL13b and LtSPL13b).In contrast, the 17th and 18th sites of the SBP domain in G3 members (LcSPL9 and LtSPL9, LcSPL15 and LtSPL15) contained Tyrosine (Y) and Cysteine (C) residues, respectively.At the 40th residue of the SBP domain, K and R residues were identified in G8 (LcSPL7 and LtSPL7) and G2 (LcSPL2 and LtSPL2, LcSPL11 and LtSPL11) members, respectively.Additionally, a G residue was found at the 15th residue in G8 members (LcSPL7 and LtSPL7), while the other residues were Serine (S).

Syntenic analysis of SPLs in Liriodendron
The chromosomal location plot of the 35 SPL genes in Liriodendron revealed a non-uniform distribution across the 19 chromosomes (Fig. 4).Notably, both LcSPLs and LtSPLs showed segmental duplicates, with three pairs of duplicated genes identified in each species (LcSPL9 and LcSPL15, LcSPL13a and LcSPL13b, LcSPL14a and LcSPL14b for L. chinense; LtSPL9 and LtSPL15, LtSPL13a and LtSPL13b, LtSPL14a and LtSPL14b for L. tulipifera).However, no tandem duplications were observed in either species.Additionally, syntenic analysis indicated that, except for LtSPL6, all 17 LtSPLs exhibited collinear relationships with the LcSPLs (Fig. 5).The absence of syntenic homology for LtSPL6 with L. chinense might be attributed to incomplete assembly of the L. chinense genome.
In addition, to infer the evolutionary relationships of SPLs, we conducted synteny analysis across four dicotyledonous plants: L. chinense, L. tulipifera, A. thaliana and Vitis vinifera.The findings revealed homology between SPL genes in Liriodendron and those in A. thaliana and V. vinifera.In particular, the highest level of collinearity was observed between L. chinense and V. vinifera, with a total of 12 pairs of genes exhibiting collinear relationships (Fig. 6).Notably, LtSPL6 exhibited collinear relationships with V. vinifera but not with A. thaliana.Overall, our results indicate a closer evolutionary affinity between SPLs from Liriodendron and V. vinifera compared with A. thaliana.This suggests that SPLs share a more recent common ancestor V. vinifera than with A. thaliana.These results support the notion that the SPLs

Identification of cis-acting element of LcSPLs and LtSPLs
To elucidate the mechanisms of transcriptional regulation, we conducted an analysis of the cis-acting elements present in the promoters of LcSPLs and LtSPLs.A total of 57 types of cis-elements were identified from the 35 SPL genes in Liriodendron (Table 1).Based on their putative functions, these elements were categorized into eight groups: site-binding-related elements, promoter-related elements, light-responsive elements, hormone-responsive elements, environmental stress-related elements and development-related elements (Fig. 7).
The most prevalent cis-elements identified were the TATA box and CAAT box, both of which were present in all LcSPLs and LtSPLs.Moreover, light-responsive elements constituted the second largest group and were found in similar numbers as hormone-responsive elements within the promoter regions of all SPL genes.This noteworthy observation suggests that both LcSPLs and LtSPLs are intricately regulated in response to diverse environmental and developmental cues.Consequently, these findings reinforce the essential roles of LcSPLs and LtSPLs in governing critical physiological processes and coordinating complex developmental programs.Importantly, the distribution patterns of promoter elements in both SPL gene clades mirrored their homology distribution, underscoring the significance of these elements in gene regulation while highlighting their evolutionary conservation.

Expression patterns of the LcSPL genes in different L. chinense tissues
The distinct functions of genes in various plant tissues are often revealed through their unique expression patterns.To gain insights into the developmental roles of LcSPLs, we quantified the expression levels of the 17 LcSPLs across 8 tissues/organs of L. chinense, including root, stem, leaf, calyx, floral bud, petal, pistil and stamen (Fig. 8).Our RT-qPCR results unveiled diverse expression patterns of LcSPLs among these tissues/organs, with specific genes exhibiting tissue-specific expression profiles.Notably, homologous genes LcSPL9 and LcSPL10 were highly expressed in flower buds and pistils.Furthermore, LcSPL2 showed the highest relative expression in pistils, with levels 30 times higher than those in the root, followed by leaves with a 24-fold increase, and approximately 15-fold higher in the sepal and flower bud compared with the root.Additionally, LcSPL7 exhibited significantly higher expression in stem tips and sepals than in other tissues.Interestingly, several

Ectopic expression of LcSPL2 in A. thaliana
To investigate the impact of LcSPL2 on flowering, we introduced its ectopic expression under the 35S CaMV promoter (35S::LcSPL2) in A. thaliana Columbia ecotype (Col-0) (WT), as depicted in Fig. 9.The base sequence of LcSPL2 can be found in Supplementary Material 1.We measured the time taken from seed sowing to the first flowering of the 35S::LcSPL2 transgenic plants under long-day (LD) conditions.Statistical analysis revealed a significant difference in the first flowering time between 35S::LcSPL2 and WT A. thaliana, with the 35S::LcSPL2 plants flowering significantly earlier.Furthermore, a reduction in the number of rosette leaves was observed in 35S::LcSPL2 compared with WT A. thaliana (Fig. 9).
To investigate whether the expression of LcSPL2 in the 35S::LcSPL2 transgenic A. thaliana influenced the expression of flowering-related genes, we performed RT-qPCR assays when the transgenic A. thaliana bloomed its first flower.Comparative analysis with WT plants revealed a significant increase in the expression of AtSOC1, AtFT, AtFUL and AtAP1 genes in the 35S::LcSPL2 transgenic plants (Fig. 10).
These results suggest that the ectopic expression of LcSPL2 in A. thaliana accelerates flowering and influences the expression of key flowering-related genes, providing valuable insights into the potential regulatory role of LcSPL2 in the flowering process.

Discussion
SPL genes are plant-specific TFs found across a wide range of green plants, including monocotyledonous, dicotyledonous and algal species (Cardon et al. 1999;Lannenpaa et al. 2004;Riese et al. 2008;Yang et al. 2008;Salinas et al. 2012).These genes play pivotal roles in regulating plant flower development and are involved in various plant growth and developmental processes, particularly in flowering regulation (Zhang et al. 2015;Han et al. 2016;Pan et al. 2017).In this study, we conducted a genome-wide analysis of the SPL genes in two Liriodendron species, L. chinense and L. tulipifera.Generally, SPL genes are classified into six to nine clades (Preston and Hileman 2013).The phylogenetic tree in this study showed that LcSPLs and LtSPLs clustered into nine clades.Paired homologous pairs of L. chinense and L. tulipifera SPLs were observed in each clade, with the exception of the G1 LtSPL6 of L. tulipifera.Furthermore, we found that the LcSPLs of L. chinense exhibited a high degree of covariance with the LtSPLs in L. tulipifera.However, both species SPLs lacked tandem duplications.Previous studies have reported wholegenome duplication events in Liriodendron (Chen et al. 2018), suggesting that the differentiation of SPL genes in Liriodendron occurred before the divergence of L. chinense and L. tulipifera.Furthermore, we found that all groups contained SPL proteins from both monocots and dicots, indicating that SPLs existed prior to the divergence of dicots and monocots and subsequently evolved independently.
The SBP structural domains of both LcSPLs and LtSPLs exhibit highly conserved sequences, including CQQC, SCR, NLS, Zn-1 and Zn-2.This conservation is consistent with other plant species (Yang et al. 2008;Zhu et al. 2020).A total  of 10 conserved motifs were identified among the SPL proteins.Interestingly, all 10 motifs were present in the G5 group, which also includes AtSPL1/12 and exhibits similar motif distribution in tobacco (De Paola et al. 2023).Considering the high degree of sequence homology, along with similar conserved structural domain features and motif distributions among the same group of LcSPLs, these genes classified in the same group may have similar biological functions.
To gain further insights into the functions of SPL genes in L. chinense, we examined the expression levels of 17 LcSPLs in eight tissues.Generally, SPLs within the same clade share similar functions.Previous studies have indicated that SPL genes located in the same clade are involved in regulating flowering time in various plants like A. thaliana, rice and alfalfa (Medicago sativa) (Martin et al. 2010;Wang et al. 2012;Gao et al. 2018b).Notably, AtSPL9/15 plays a crucial role in phase transition, anthocyanin accumulation and flowering regulation (Wang et al. 2009;Gou et al. 2011).In this study, we observed collinear relationships between LcSPL9/15 and AtSPL9/15.Moreover, LcSPL9/15 exhibited significantly elevated expression levels in flower buds and pistils.Therefore, further investigations are warranted to unravel the regulatory function of LcSPL9/15 in flowering control in L. chinense.Understanding the role of LcSPL9/15 could provide valuable insights into the intricate mechanisms governing flowering regulation in this species.Additionally, both LcSPL2/11 genes showed high expression in leaves and pistils.Interestingly, three AtSPL genes (AtSPL2/10/11), from the same group as LcSPL2/11, have been previously identified as regulators of flowering time in A. thaliana (Yao et al. 2019;Ma et al. 2020).This finding indicates that LcSPL2/11 may also be involved in the regulation of flowering time in L. chinense.Further investigations are needed to elucidate the specific roles of LcSPL2/11 in the flowering process of this species.
Previous studies have conducted functional overexpression or deletion experiments on SPL genes in various plant species.For instance, in Fortunella hindsii, overexpression of three FhSPLs in A. thaliana resulted in early flowering phenotypes (Li et al. 2023).Similarly, the AtSPL3/4/5 homologs from Platanus acerifolia induced early flowering when expressed in A. thaliana (Han et al. 2016).In alfalfa, MsSPL13 and MsSPL20 were found to regulate the vegetative growth phase.Plants overexpressing these SPL genes exhibit increased biomass and delayed flowering time (Gao et al. 2018b;Ma et al. 2022).In our study, we employed an ectopic expression system to investigate the function of LcSPL2.The transgenic A. thaliana plants with 35S::LcSPL2 showed an early-flowering phenotype (Fig. 8), with no significant changes in flower morphology, as anticipated.
As the primary effectors in the age-flowering pathway, SPL genes play a crucial role in regulating flowering time through two main mechanisms (Teotia and Tang 2015).Firstly, they operate within the microRNA regulatory network by activating downstream miR172, which, in turn, inhibits the activity of the AP2s and releases the activity of SOC1 and other genes to promote flowering (Yant et al. 2010).Secondly, SPL genes directly bind to the promoters of FT, SOC1, AP1 and FUL, leading to the promotion of flowering in plants (Wang et al. 2009;Yamaguchi et al. 2009;Kim et al. 2012).In this study, we observed a significant increase in the expression of AtSOC1, AtFT, AtFUL and AtAP1 in 35S::LcSPL2 transgenic plants.This suggests that ectopic-transformed LcSPL2 promotes flowering by increasing the expression levels of flowering-related genes in A. thaliana.However, further investigation is needed to elucidate its regulatory mechanism with genes such as FT and SOC1 in L. chinense.Additional studies will be required to unravel the complex regulatory network underlying flowering in L. chinense.

Conclusion
This study comprehensively analyses the SPL gene family in L. chinense and L. tulipifera.Seventeen LcSPL genes and 18 LtSPL genes were identified and divided into 9 groups.Tissue expression analysis showed that LcSPLs were expressed more in flower-related tissues.Overexpression of LcSPL2 in A. thaliana accelerates flowering under LD conditions.Our study lays a cornerstone for further understanding the functions of LcSPL genes.

Figure 2 .
Figure 2. Phylogenetic relationships and architecture of the conserved motifs in SPL proteins from Liriodendron.(A) The phylogenetic tree was constructed based on the full-length sequences of LcSPL and LtSPL proteins.(B) Conserved domains structure of LcSPL and LtSPL genes.Black lines indicate relative protein lengths, boxes represent conserved domains and domains are colour-coded.(C) Amino acid motifs in the LcSPL and LtSPL proteins are represented by coloured boxes.Black lines indicate relative protein lengths.

Figure 3 .
Figure 3. Multiple sequence alignment of SBP domain between LcSPLs and LtSPLs proteins.C3H and C2HC were two zinc finger structures, NLS was bidirectional nuclear localization signal, which were corresponding to the sequence logos of conserved domains.

Figure 4 .
Figure 4. Intraspecific synteny analysis of LcSPLs and LtSPL.(A) Intraspecific synteny analysis of SPL genes in L. tulipifera.The chromatic line indicates there is a collinearity between two given LtSPL genes.(B) Intraspecific synteny analysis of SPL genes in L. chinense.The chromatic line indicates there is a collinearity between two given LcSPL genes.

Figure 5 .
Figure 5. Synteny analysis of SPL genes from LcSPLs and LtSPLs.Grey lines in the background indicate collinear blocks between L. chinense and L. tulipifera, whereas the chromatic lines highlight syntenic SPL gene pairs.

Figure 6 .
Figure 6.Synteny analysis of SPL genes among L. chinense, L. tulipifera, A. thaliana and V. vinifera.Grey lines in the background indicate the collinear blocks within different plant genomes, while colour lines highlight the syntenic SPL gene pairs.

Figure 7 .
Figure 7. Cis-element regions of SPL genes promoters were analysed by PlantCARE in L. chinense and L. tulipifera.The numbers in the box represent the number of cis-element.

Figure 8 .
Figure 8. Expression analysis of LcSPLs in root, stem, leaf, calyx, floral bud, petal, pistil and stamen of L. chinense.The expression was determined by quantitative RT-PCR.LcActin97 was used as an internal control.Means with different letters were significantly different.The error bars indicated ± SD of three technical replicates.Analysis of variance (ANOVA) with a subsequent Duncan's test was performed (P < 0.05).

Figure 9 .
Figure 9.The phenotypes and relative expression levels of LcSPL2 in the transgenic overexpression lines and WT A. thaliana.(A) The rosette leaf of WT A. thaliana and 35S::LcSPL2-1 A. thaliana, bar = 1 cm.(B) The right three A. thaliana plants represented three transgenic lines, LcSPL2-1, LcSPL2-3 and LcSPL2-4, bar = 1 cm.(C) Comparison of the number of rosette leaf at flowering between each 35S::LcSPL2 line and WT A. thaliana.(D) Comparison of the flowering time in each 35S::LcSPL2 transgenic line and WT A. thaliana.The first flowering duration was recorded in days from seeding to the occurrence of the first flower, unit in days.The error bars indicate the standard deviation (SD) of three biological replicates consisting of independent samples (no less than 10 plants per replicate).ANOVA with a subsequent Duncan's test was performed (double asterisks, P < 0.01).

Figure 10 .
Figure 10.RT-qPCR analysis of four genes in WT A. thaliana and three 35S::LcSPL2 overexpression lines under LD condition.(A)-(D) RT-qPCR analysis of four flowering-related genes, AtSOC1, AtFT, AtFUL and AtAP1, in WT and transgenic A. thaliana.Samples were collected on the days to the initial flowering of 35S::LcSPL2 and WT A. thaliana.AtActin2 was used as an internal control.The error bars indicate ± SE of three technical replicates.Means with different letters were significantly different.ANOVA with a subsequent Duncan's test was performed (P < 0.05).

Table 1 .
Basic information about LcSPLs

Table 2 .
Basic information about LtSPLs

ID CDS length (bp) Protein length (aa) pI Mw (Da) Exon Number
Furthermore, Table1provides detailed characteristics of each individual SPL gene, including isoelectric point (pI) and the number of predicted exons.